Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, United States of America.
PLoS One. 2009 Sep 29;4(9):e7253. doi: 10.1371/journal.pone.0007253.
BtubA and BtubB are two tubulin-like genes found in the bacterium Prosthecobacter. Our work and a previous crystal structure suggest that BtubB corresponds to alpha-tubulin and BtubA to beta-tubulin. A 1:1 mixture of the two proteins assembles into tubulin-like protofilaments, which further aggregate into pairs and bundles. The proteins also form a BtubA/B heterodimer, which appears to be a repeating subunit in the protofilament.
METHODOLOGY/PRINCIPAL FINDINGS: We have designed point mutations to disrupt the longitudinal interfaces bonding subunits into protofilaments. The mutants are in two classes, within dimers and between dimers. We have characterized one mutant of each class for BtubA and BtubB. When mixed 1:1 with a wild type partner, none of the mutants were capable of assembly. An excess of between-dimer mutants could depolymerize preformed wild type polymers, while within-dimer mutants had no activity.
An essential first step in assembly of BtubA + BtubB is formation of a heterodimer. An excess of between-dimer mutants depolymerize wild type BtubA/B by sequestering the partner wild type subunit into inactive dimers. Within-dimer mutants cannot form dimers and have no activity.
BtubA 和 BtubB 是在 Prosthecobacter 细菌中发现的两个微管蛋白样基因。我们的工作和以前的晶体结构表明，BtubB 对应于 alpha-微管蛋白，BtubA 对应于 beta-微管蛋白。两种蛋白质以 1:1 的比例组装成微管蛋白样原纤维，这些原纤维进一步聚集形成对和束。这些蛋白质还形成 BtubA/B 异二聚体，它似乎是原纤维中的重复亚基。
方法/主要发现：我们设计了点突变来破坏将亚基结合成原纤维的纵向界面。突变体分为两类，在二聚体内部和二聚体之间。我们对每种类型的一个突变体进行了 BtubA 和 BtubB 的特征描述。当与野生型伙伴以 1:1 的比例混合时，没有一个突变体能够组装。过量的二聚体突变体可以解聚预先形成的野生型聚合物，而二聚体内部突变体没有活性。
BtubA+BtubB 组装的一个基本的第一步是形成异二聚体。过量的二聚体突变体通过将野生型亚基隔离到无活性的二聚体中来解聚野生型 BtubA/B。二聚体内部突变体不能形成二聚体，也没有活性。
